The etiologies of the two major inflammatory bowel diseases (IBD), Crohn's disease and ulcerative colitis, remain unknown, although it seems clear that both diseases can be influenced by environmental and genetic factors which contribute to initiation, amplification and prolongation of the inflammatory response. The most commonly prescribed drugs for IBD therapy are those which generate high concentrations of 5-aminosalicylic acid (5-ASA) in the colonic lumen. Despite known efficacy of these drugs for treatment of active disease and maintenance of quiescent disease, therapeutic mechanisms remain uncertain, due to uncertainty of the site of action of 5-ASA in the colonic mucosa. The Montrose group at Johns Hopkins University has developed ratiometric microscopy methods to image 5-ASA and its major metabolite (N-acetyl-5-ASA; Ac-5-ASA) simultaneously in living mouse colonic mucosa and we have extended this technique by the use of multiphoton microscopy.